This invention claims priority of the German patent application 100 43 992.6 which is incorporated by reference herein.
The present invention concerns a method for examining a specimen by means of a confocal scanning microscope.
A method for examining a specimen by means of a scanning microscope, and a confocal scanning microscope, of the kinds cited above are known from practical use. In known scanning microscopy, a specimen is illuminated with an illuminating light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of the illuminating light beam is generally moved in one specimen plane by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. The tilting of the mirrors that substantially constitute the beam deflection device is brought about, for example, with the aid of galvanometer positioning elements, both fast resonant galvanometers as well as slower and more accurate non-resonant galvanometers being used. The power of the light coming from the specimen is measured as a function of the position of the scanning beam or illuminating light beam.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of an illuminating light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the light source is focused onto a pinhole, a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and detectors for detecting the reflected or fluorescent light. The illuminating light or illuminating light beam must usually be coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen passes, in the most commonly used descan arrangement, via the same scanning mirrors or the same beam deflection device back to the beam splitter and passes through the latter, then being focused onto the detection pinhole behind which the detectors (usually photomultipliers) are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop; what is obtained is a point datum that results, by way of sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
At present, specimens are usually illuminated over the entire scan field with light of one wavelength, or simultaneously with light of several wavelengths. For this reason, comparative examinations whose purpose is to examine specimens under different spectral illumination conditions but under otherwise identical boundary conditions are performed sequentially on one specimen or sequentially on identically prepared specimens.
In the case of examinations based on fluorescence resonance energy transfer (FRET), molecules are excited optically, for example with light at the 488 nm wavelength. The emitted light of these so-called donor molecules, which in the present example would have a wavelength of approx. 543 nm, results, by way of so-called Fxc3x6rster transfer, in the excitation of other closely adjacent molecules (acceptor molecules). The latter then emit light at a wavelength of approx. 570 nm. At present, to control specimen preparation for experiments based on fluorescence resonance energy transfer (FRET), test measurements are performed before the actual examinations are made. In the present example irradiation would first be applied with light at a wavelength of 534 nm, in order to excite the acceptor molecules directly and in order to acquire an image at the detection wavelength of 570 nm. The specimen would then be displaced mechanically, for example with an X-Y stage, and the xe2x80x9cactualxe2x80x9d examination would be performed at a different point using excitation light at the 488 nm wavelength.
In order to rule out direct excitation of the acceptor with the light that is actually intended for excitation of the donor (in this example, 488 nm), the bleaching behavior of the acceptor and donor can be measured in direct excitation. From a comparison between the bleaching coefficients with direct excitation and those with FRET excitation, conclusions can be drawn as to the degree of direct excitation.
Ideally, the track of the deflected illuminating light beam on the specimen surfacexe2x80x94or, in the case of a confocal arrangement, in a layer plane in the specimenxe2x80x94should describe a meander. This involves first scanning a line in the X direction at a constant Y position, then a Y displacement with no change in X position, and then scanning a line in the negative X position at a constant Y position. In reality, because of the inertia of the moving galvanometer components and the mirrors of the beam deflection device, a meander shape of this kind can be approximately achieved only for low scanning rates. At reasonable scanning rates of more than 100 Hz, the scanning track of the illuminating light beam actually describes a sine-like curve, which creates the need for correction of the resulting deviations from the ideal situation. For example, the track speed in the vicinity of the reversal points is lower than in the linear sine region, resulting (inter alia) in greater bleaching in those regions. It has therefore been usual for some time to interrupt the specimen illumination while passing through the reversing portions, using mechanical stops that limit the image field or by means of suitable optical arrangementsxe2x80x94for example with acoustooptical modulators (AOTFs). This technique of interrupting the beam during scanning is called xe2x80x9cblanking.xe2x80x9d An arrangement with mechanical stops was incorporated as early as 1990 in a confocal laser scanning microscope of the applicant. An arrangement having an acoustooptical modulator is described in Scientific and Technical Information Vol. XI, No. 1, pp. 9-19, June 1995, xe2x80x9cLeica TCS 4D UVxe2x80x94The system concept for Multiparameter Confocal Microscopy.xe2x80x9d This document explains the sine-like trajectory and the problems associated with it, although blanking is not explicitly mentioned. Controlled bleaching-out of any desired predefinable specimen regions using an AOTF arrangement, which makes it possible to illuminate various regions of a specimen with different light intensities, is described in P. Wedekind et al., xe2x80x9cScanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging,xe2x80x9d Journal of Microscopy, Vol. 176, Part 1, October 1994, pp. 23-33. This document illustrates a blanking technique at a very high technical level.
Unexamined Patent Application DE 198 29 981 of Carl Zeiss Jena GmbH, xe2x80x9cMethod and arrangement for confocal microscopy,xe2x80x9d describes the elimination of the bleaching problem, and additionally the elimination of bleed-through, by the fact that the spectral composition and/or the intensity of the laser light coupled into the microscope beam path is modified while deflection continues without interruption; as a result, at least two adjacent locations or scan points of the specimen are impinged upon by light of differing spectral properties and/or different intensity.
A problem with the known method and the known confocal scanning microscope is that it is not clear how a detail of a specimen that is to be evaluated can be selected for differentiated illumination. Reliable selection and definition of the details of interest in the specimen is therefore not possible.
It is therefore an object of the present invention to provide a method for examining a specimen, which enables a user to select specific regions of interest and limit information collection to the selected regions.
The present invention provides a for examining a specimen by means of a confocal scanning microscope comprising the steps of:
generating an illuminating light beam with at least one light source,
deflecting, with a beam deflection device, the illuminating light beam over a specimen,
acquiring a preview image;
marking of at least one region of interest in the preview image;
allocating individual illuminating light beam wavelengths or illuminating light beam power levels to the at least one region of interest;
illuminating at least one region of the specimen in accordance with the allocation, the at least one region of the specimen corresponding to the at least one region of interest in the preview image, wherein the illuminating light beam is guided such that substantially only the at least one region of the specimen is illuminated, and
detecting reflected and fluorescent light proceeding from the at least one region of specimen.
First a preview image is acquired. This supplies to the observer a visual depiction of the specimen being examined. Marking of at least one region of interest in the preview image is then accomplished. These two method steps make possible, in a manner according to the present invention, particularly simple selection and definition of a detail of interest of a specimen. The observer simply needs to study the preview image in order then to make a marking in the preview image.
This is then followed by an allocation of individual illuminating light beam wavelengths and/or illuminating light beam power levels to the region or regions. The region or regions of the specimen is or are then illuminated in accordance with the allocation, followed by detection of the reflected and/or fluorescent light proceeding from the specimen. This completes the examination method. In this context, quite individually selected regions can be illuminated.
In particularly simple fashion, the beam deflection device could comprise galvanometer positioning elements. Galvanometer positioning elements of this kind could preferably be controllable by way of a computer, with which the beam deflection speeds can be adapted individually to requirements in terms of the marked region or regions.